Sila Konur Ultanir: Projects

Hippo signaling in neuronal development

Neuron specific deletion of hippo (Mammalian Ste20-like kinase MST1/2 in mammals) or wts (Large Tumour Suppressor kinase Lats1/2 in mammals) in Drosophila sensory neurons leads to deficiencies in maintenance of neuronal dendritic arbors, while deletion of hippo or another NDR kinase trc (Nuclear Dbf2 Related kinase, NDR1/2 in mammals) causes increased dendrite branching in flies, implicating MST1/2 signaling in dendritic development. Although roles of the tumour suppressor Hippo/ MST1/2 in cell division, apoptosis, cell polarity and tissue size control have been studies in detail, whether or not MST1/2 functions in mammalian neuronal development is an open question. We plan to address this question using MST1/2 neuronal conditional knockout mice.

MST1 and MST2 are expressed during development in various brain regions. In situ hybridisation revealed that MST1 is localised to hippocampal pyramidal cell layers and dentate granule cells. In order to test if MST1/2 function is required in neuronal development, we have crossed Mst1/2 floxed (Mst1 f/f; Mst2 f/f) mice with Nex-Cre mice, which express Cre recombinase in excitatory neurons of hippocampus and cortex.

As expected, MST1 and MST2 protein levels are reduced in the knockout hippocampi. In these neuronal conditional MST1/2 knockout mice we are investigating if MST1/2 is required for synapse development by performing whole-cell patch clamp electrophysiological recordings. We are also characterising dendrite arbors and dendritic spine morphologies using either fluorescently-recovered biocytin fills obtained during recordings or by using Thy1YFP mice and confocal imaging.

We are then going to characterise if MST1/2 acts via its canonical effector Lats1/2 or a related kinase NDR1/2. In parallel, we aim to identify novel MST1/2 kinase substrates by using chemical genetics.

Role of CDKL5 in brain development.

Cyclin-dependent kinase like 5 (Cdkl5) also known as serine threonine kinase 9 (Stk9) is a member of cyclin-dependent kinase family of serine/threonine protein kinases. Mutations in cdkl5 gene have been associated with severe neurodevelopmental disorders such as Infantile Spasms, West Syndrome and atypical Rett syndrome. Cdkl5 gene is on the X chromosome and mutations in cdkl5 affects girls.

Although rapidly growing evidence implies cdkl5 in these developmental disorders, the current understanding of the biological function of CDKL5 is limited. CDKL5 is heavily expressed in the brain during development and in adult. In cortical neurons CDKL5 shuttles between nucleus and cytoplasm and loss of CDKL5 function causes reduced dendrite arborisation. The function of cdkl5 in synaptic development and dendritic spine morphogenesis has not been investigated. More importantly the molecular mechanisms by which CDKL5 exert its functions is unknown.

A direct and unbiased screen to identify the phosphorylation targets of CDKL5 would address its molecular functions. We propose to use our kinase substrate identification method to reveal the substrates of CDKL5 and the phosphorylation sites. Our next goal is to investigate the significance of CDKL5 and its substrates in neuronal development using morphological and physiological methods in cultures and using mouse models.